Assessment of antimalarial activity of crude extract of Chan-Ta-Lee-La and Pra-Sa-Chan-Dang formulations and their plant ingredients for new drug candidates of malaria treatment: In vitro and in vivo experiments

The emergence and spread of antimalarial drug resistance have become a significant problem worldwide. The search for natural products to develop novel antimalarial drugs is challenging. Therefore, this study aimed to assess the antimalarial and toxicological effects of Chan-Ta-Lee-La (CTLL) and Pra-Sa-Chan-Dang (PSCD) formulations and their plant ingredients. The crude extracts of CTLL and PSCD formulations and their plant ingredients were evaluated for in vitro antimalarial activity using Plasmodium lactate dehydrogenase enzyme and toxicity to Vero and HepG2 cells using the tetrazolium salt method. An extract from the CTLL and PSCD formulations exhibiting the highest selectivity index value was selected for further investigation using Peter’s 4-day suppressive test, curative test, prophylactic test, and acute oral toxicity in mice. The phytochemical constituents were characterized using gas chromatography-mass spectrometry (GC-MS). Results showed that ethanolic extracts of CTLL and PSCD formulations possessed high antimalarial activity (half maximal inhibitory concentration = 4.88, and 4.19 g/mL, respectively) with low cytotoxicity. Ethanolic extracts of the CTLL and PSCD formulations demonstrated a significant dose-dependent decrease in parasitemia in mice. The ethanolic CTLL extract showed the greatest suppressive effect after 4 days of suppressive (89.80%) and curative (35.94%) testing at a dose of 600 mg/kg. Moreover, ethanolic PSCD extract showed the highest suppressive effect in the prophylactic test (65.82%) at a dose of 600 mg/kg. There was no acute toxicity in mice treated with ethanolic CTLL and PSCD extracts at 2,000 mg/kg bodyweight. GC-MS analysis revealed that the most abundant compounds in the ethanolic CTLL extract were linderol, isoborneol, eudesmol, linoleic acid, and oleic acid, whereas ethyl 4-methoxycinnamate was the most commonly found compound in the ethanolic PSCD extract, followed by 3-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4H-chromen-4-one, flamenol, oleic acid amide, linoleic acid, and oleic acid. In conclusions, ethanolic CTLL and PSCD extracts exhibited high antimalarial efficacy in vitro. The ethanolic CTLL extract at a dose of 600 mg/kg exhibited the highest antimalarial activity in the 4-day suppressive and curative tests, whereas the ethanolic PSCD extract at a dose of 600 mg/kg showed the highest antimalarial activity in the prophylactic test.


Reviewer #2:
• The authors mentioned that "Each plant and formulation 165 were extracted using ethanol and distilled water.".What was the percentage of the used ethanol?
Response: The percentage of ethanol at 95% has been added to the manuscript.Please see page 8, line 165.
• Please mention the exact temperature where room temperature varies from country to country "at room temperature for 72 h…..".
Response: Room temperature at 25 °C has been added to the manuscript.Please see page 9, line 169.
• Please mention the source of Plasmodium falciparum.

Response:
The sentence about the source of Plasmodium falciparum has been added to the manuscript.Please see page 9, lines 182-184.
• For parasitemia determination "The percentage of parasitemia was determined daily using a light microscope": I suggest adding a reference (cite) and calculate the percentage of suppression from The antiplasmodial and spleen protective role of crude Indigofera oblongifolia leaf extract traditionally used in the treatment of malaria in Saudi Arabia.Drug Des Devel Ther.2015 Nov 25;9:6235-46.doi: 10.2147/DDDT.S94673.
Response: A suggested reference (cite) has been added.Please see page 9, line 190.
• If the used mice were adult (6-8 weeks) add adult before mice.

Response:
The word "adult" has been added to the manuscript.Please see page 13, line 268.
• Mention how you have calculated 1× 10 7 P. berghei-parasitized RBCs to be the used dose.
Mention the used chamber.
Response: For the calculation of 1× 10 7 P. berghei-parasitized RBCs to be the used dose, we calculated from the level of parasitemia in donor mice.When the level of parasitemia in donor mice reached 20-30%, the donor mouse was subsequently sacrificed and blood collected (via cardiac puncture) into a heparinized tube.Based on the parasitemia level of the donor mouse and the red blood cell (RBC) count of a normal mouse, the collected blood was diluted with normal saline (0.9%), so that 1 ml of blood contains 5 x 10 7 infected RBCs.Each mouse was injected with 0.2 ml of blood containing 1 x 10 7 P. berghei-infected RBCs via the intraperitoneal route.Response: For the infection of mice, we used whole blood.Briefly, blood from donor mice was collected and then diluted with 0.9% physiological isotonic saline before injected into mice in the experiment.
• In table 10: "Table 10.Percent suppression of the ethanolic extract of the CTLL and PSCD formulations in prophylactic test" mention the day of blood collection.Why this day.

Response:
• The word "Day 6" has been added to the table name.Please see page 30, line 563.
• We monitored parasitemia on day 6 (day 6 after mice received the extract or day 3 postinfection) because the parasitemia level increased steadily beginning on day 3 post-infection.
The reason could be that only approximately one-third of infected red blood cells are able to circulate freely in the blood circulation within 48 hours, whereas the remaining parasites stick to the venules and capillaries.Therefore, blood collection at day 3 post-infection would provide a precise assessment of the parasitemia.
Ref: Nwonuma CO, Balogun EA, Gyebi GA.Evaluation of antimalarial activity of ethanolic extract of Annona muricata L.: An in vivo and an in-silico approach.Journal of Evidence-Based Integrative Medicine.2023;28.• In the histopathology: I cannot see any histopathological change with this magnification.I found no figure caption describing the groups and the histopathological change.I cannot see the scale bar.I also suggest in addition to add a higher magnification to add a histology score.Response: Thank you for your suggestion.A new figure of histopathological change with a higher magnification has been added.• I suggest comparing your results with authors used any of the used extracts or mention the activity of other extracts and write on the important of using medithinal plants against malaria.You can use "Medicinal plants as a fight against murine blood-stage malaria.Saudi J Biol Sci.2021 Mar;28(3):1723-1738. doi: 10.1016/j.sjbs.2020.12.014.Response: Thank you for your valuable comment.I have already compared results with authors who used any of the used extracts or mentioned the activity of other extracts (please see pages 37-38, lines 720-723) and wrote about the importance of using medicinal plants against malaria (please see pages 34, lines 63-63).